Previous indirect studies have suggested that activation of phospholipase A2 (PLA2) is a pivotal step in hormone release, in particular that of insulin. However, there has been no specific demonstration of the presence of a PLA2 in intact islets and its activation by relevant islet stimuli such as D-glucose. Furthermore, most studies have focused on one hydrolytic by-product of PLA2 action, arachidonic acid (AA) and the contribution of lipoxygenase-derived metabolites of AA such as 12-hydroperoxyeicosatetraenoic acid (12-HPETE) to insulin (I) release; the role of the second phospholipid moiety generated by PLA2 action (lysophospholipids; lyso-PLs) remains virtually unexplored in any endocrine cell system. Our preliminary data strongly implicate lyso-PLs in the initiation of glucose-induced I release, with the co-generation of AA metabolites serving to modulate this response. This protocol, therefore, seeks to measure specific PLA2 activity (and its response to islet agonists, such as glucose; inhibitors, such as epinephrine; or pathophysiologic perturbations such as starvation or chemically-induced diabetes) in intact adult rat islets by the simultaneous determination of lyso-PL and 12-HPETE accumulation using high performance liquid chromatographic analysis of membrane extracts or media from islets. Islets will be pre-labelled with 3H-AA, 14C-stearate, (32P)-Pi and/or 3H-glycerol in order to label, respectively, the sn-2 carbon, the sn-1-carbon, the polar head groups, and the 3-carbon backbone of phospholipids. The accumulation of various phospholipids and lysophospholipids will be monitored by HPLC after exposure of the islets to D-glucose, exogenous PLA2 and other test compounds in the presence or absence of p-hydroxymercuribenzoate (to impede the removal of any lyso-PL generated). I secretion is assessed, in parallel, in static incubations of intact rat islets, and in perifusions of intact islets and of monolayer-cultured, dispersed neonatal islet cells. We will examine the time- and dose-correspondence between I release and lyso-PL formation in response to various perturbations of islet secretion, and will assess the stereo- and anomeric specificity, Ca++ dependence and dependence on fuel flux of the phospholipid response to glucose. In turn, the mechanism of lyso-PL stimulation of I release will be assessed by examining its response to relatively specific pharmacologic perturbations and through measurements of relevant parameters such as cyclic AMP or intracellular Ca++ accumulation. We will assess whether exogenous lyso-PLs or exogenous PLA2 can reverse the blockade of glucose-induced I release caused by phospholipase inhibitors, fasting, chemically-induced diabetes, epinephrine or somatostatin. These studies should define the presence of, and basic mechanisms involved in, regulating islet PLA2 activity. These studies may provide insights into stimulus-secretion coupling in general and specifically into the insulin secretory defect characteristic of human diabetes mellitus.